Genotyping Approaches for Identification and Characterization of Staphylococcus aureus

Genotyping methods are vital epidemiological tools for discriminating different bacterial isolates within same species, which in turn provide useful data in tracing source of infection and disease management. There have been a revolutionary efforts in ways to distinguish between bacterial types and subtypes at molecular level utilizing DNA in the genomes. Notably, the growth of various DNA typing methods has provided innovative apparatuses for improved surveillance and outbreak investigation. Thus, early identification and genotyping are indispensable as resources for managing therapeutic treatment and the control of rapid expansion of clinically important bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) has been in a great attention due to its contagious nature and subjected to various typing analyses. Thus, in this chapter, we aimed to review the contribution of various genotyping methods of commonly used as well as those unique to staphylococci in understanding its epidemiology, infection and dissemination pattern, and to provide an impression of specific advantages and disadvantages of each tool

The Evolution and Dissemination of Methicillin Resistance Determinant in Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen and is frequently associated with the antimicrobial resistance. There has been horizontal gene transfer of Staphylococcus chromosome cassette mec (SCCmec) among the staphylococcal species that colonize a similar colonization niche, which eventually results in emergence of new variant with enhanced survival ability in terms of antimicrobial resistance and virulence level in S. aureus. Evolution and dissemination of SCCmec structure resulted in the emergence of methicillin-resistant S. aureus (MRSA) clones around the world covering hospital, community, and livestock settings. MRSA also has the ability to resist different antibiotic profiles known as multidrug-resistant S. aureus (MDR S. aureus)

Antimicrobial activities of plant extracts against methicillin-susceptible and methicillin-resistant Staphylococcus aureus

The present study was carried out to compare the antimicrobial activities of methanol leaf extracts of Bauhinia purpurea, Dicranopterislinearis, Melastomamalabathricum and Muntingiacalabura portrayed by different antimicrobial assays against methicillin-susceptible and methicillin- resistant Staphylococcus aureus strains. Antimicrobial activities of the methanol leaf extracts were preliminarily screened by disc diffusion. Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution and colorimetric assay (resazurin). Based on disc diffusion method, S. aureusATCC®700699™ (MRSA) elucidated higher susceptibility pattern against all plant extracts compared to S. aureusATCC®25923™ (MSSA). Taking results from all employed assays into consideration, M. calaburamethanol leaf extract comparably elicited the highest antimicrobial activity than the other methanol leaf extracts against both microorganisms. The MIC values were determined by colorimetric assay (resazurin) due to pigmentations of the methanol leaf extracts that obscured visual growth turbidity inspection. Complication in colour changes observation in colorimetric assay to determine MBC was overcome by employing the conventional plating method. This study suggested that all antimicrobial assays should be carried out concurrently so as the data obtained can be comparatively analysed for a better outcome as each antimicrobial assay has its own shortfall

Identification of Vermamoeba vermiformis and Tetramitus sp. Isolated from the Gills of Oreochromis sp. (Red Hybrid Tilapia)

This study is the first report of an infestation of amoeba in the gills of asymptomatic Oreochromis sp. from the Manir River, Terengganu, Malaysia. The results confirmed the presence of two species of amoeba, Vermamoeba vermiformis and Tetratmitus sp., based on the 18 ribosomal RNA gene sequences with 99% sequence similarity. Morphological observations using light and scanning electron microscopy supported the findings, demonstrating both amoeba species' specific features and locomotions. In the trophozoite stage, the mean size of V. vermiformis in the locomotive form was between 25.55 µm in length and 4.17 µm in width. The mean size of Tetramitus sp. was 20.96 µm in length and 8.66 µm in width. The diameter of the cyst for V. vermiformis was 4.67 µm and Tetramitus sp. was 3.37 µm. V. vermiformis was characterized by cylindrical trophozoites and single monopodial morphology. Tetramitus sp. showed a limax-type and various cell shapes. Infestation of the amoeba was also confirmed by histopathological observation in the lamella region with distinct amoeba characteristics compared to the lamellae's epithelial tissue. These findings revealed the presence of V. vermiformis and Tetramitus sp. infestation in the gills of Oreochromis sp. and its potential pathogenic activity, whilst the symbiosis interaction in Oreochromis sp. is still unidentified

Hexavalent molybdenum reduction to mo-blue by acinetobacter calcoaceticus

A local molybdenum-reducing bacterium was isolated and tentatively identified as Acinetobacter calcoaceticus strain Dr.Y12 based on carbon utilization profiles using Biolog GN plates and 16S rDNA comparative analysis. Molybdate reduction was optimized under conditions of low dissolved oxygen (37 degrees C and pH 6.5). Of the electron donors tested, glucose, fructose, maltose and sucrose supported molybdate reduction after 1 d of incubation, glucose and fructose supporting the highest Mo-blue production. Optimum Mo-blue production was reached at 20 mmol/L molybdate and 5 mmol/L phosphate; increasing the phosphate concentrations inhibited the production. An increase in an overall absorption profiles, especially at peak maximum at 865 nm and the shoulder at 700 nm, was observed in direct correlation with the increased in Mo-blue amounts. Metal ions, such as chromium, cadmium, copper, mercury and lead (2 mmol/L final concentration) caused approximately 88, 53, 80, 100, and 20 % inhibition, respectively. Respiratory inhibitors, such as antimycin A, rotenone, sodium azide and cyanide showed in this bacterium no inhibition of the Mo-blue production, suggesting that the electron transport system is not a site of molybdate reduction

Microbiological evaluation on raw materials and food contact surfaces of ‘keropok lekor’ premises in Kuala Nerus, Terengganu and their prevalence of antibiotic resistant bacteria

‘Keropok lekor’ is a popular street food and widely available in Terengganu. However, there is limited study on the microbiological status of sources of contamination from ‘keropok lekor’ premises in Terengganu. Microbiological quality of raw materials and food contact surfaces were determined by Total Plate Count, coliform count, Staphylococcus spp. and Vibrio spp. count. The presumptive of Escherichia coli, Staphylococcus aureus and Vibrio spp. were further identified by phenotypic identification such as IMViC tests and API20 NE identification system. The antibiotic resistance of the identified bacteria was determined using disc diffusion method. Escherichia coli and Vibrio were predominant microorganisms isolated from raw materials, whereas Staphylococcus was the predominant microorganism on food contact surfaces. The boiling process of ‘keropok lekor’ at 100°C was significantly (p < 0.05) reduced the Total Plate Counts, coliform, Staphylococcus spp. and Vibrio spp. count for safe consumption. The identified antibiotic resistance of E. coli, S. aureus and Vibrio spp. showed that the bacteria were within controllable emergence strains, whereby the present antibiotics used are able to suppress the growth. Regular monitoring programme is essential to further improve the microbiological quality of raw materials and food contact surfaces

Bacterial reduction of hexavalent molybdenum to molybdenum blue.

A bacterium that was able to tolerate and reduce as high as 50 mM of sodium molybdate to molybdenum blue has been isolated from a metal recycling ground. The isolate was tentatively identified as Serratia sp. strain Dr.Y8 based on the carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. ANOVA analysis showed that isolate Dr.Y8 produced significantly higher (P < 0.05) amount of Mo-blue with 3, 5.1 and 11.3 times more molybdenum blue than previously isolated molybdenum reducers such as Serratia marcescens strain Dr.Y6, E. coli K12 and E. cloacae strain 48, respectively. Its molybdate reduction characteristics were studied in this work. Electron donor sources such as sucrose, mannitol, fructose, glucose and starch supported molybdate reduction. The optimum phosphate, pH and temperature that supported molybdate reduction were 5 mM, pH 6.0 and 37°C, respectively. The molybdenum blue produced from cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Metal ions such as chromium, silver, copper and mercury resulted in approximately 61, 57, 80, and 69% inhibition of the molybdenum-reducing activity at 1 mM, respectively. The reduction characteristics of strain Dr.Y8 suggest that it would be useful in future molybdenum bioremediation

Antiulcer activity of Muntingia calabura leaves involves the modulation of endogenous nitric oxide and nonprotein sulfhydryl compounds

Context: Muntingia calabura L. (Muntingiaceae) is a native plant species of the American continent and is widely cultivated in warm areas in Asia, including Malaysia. The plant is traditionally used to relieve pain from gastric ulcers. Objective: This study was designed to determine the antiulcer activity of a methanol extract of M. calabura leaves (MEMC) and the possible mechanisms of action involved. Materials and methods: An acute toxicity study was conducted using a single oral dose of 2000 mg/kg MEMC. The antiulcer activity of MEMC was evaluated in absolute ethanol- and indomethacin-induced gastric ulcer rat models. MEMC was administered orally (dose range 25–500 mg/kg) to rats fasted for 24 h. The animals were pretreated with NG-nitro-l-arginine methyl esters (l-NAME) or N-ethylmaleimide (NEM) prior to MEMC treatment to assess the possible involvement of endogenous nitric oxide (NO) and nonprotein sulfhydryl (NP-SH) compounds in the gastroprotective effect of MEMC. Results: As the administered dose did not cause toxicity in the rats, the oral median lethal dose (LD50) of MEMC was >2000 mg/kg in rats. MEMC exerted significant (p < 0.001) gastroprotective activity in the ethanol- and indomethacin-induced ulcer models dose-dependently. Histological evaluation supported the observed antiulcer activity of MEMC. l-NAME and NEM pretreatment significantly (p < 0.05) reversed and abolished the gastroprotective effect of MEMC, respectively. Discussion and conclusion: The results obtained indicate that MEMC has significant antiulcer activity that might involve the participation of endogenous NO and NP-SH compounds. These findings provide new pharmacological information regarding the potential use of M. calabura

Reduction of Mo(VI) by the bacterium Serratia sp. strain DRY5

The need to isolate efficient heavy metal reducers for cost effective bioremediation strategy have resulted in the isolation of a potent molybdenum-reducing bacterium. The isolate was tentatively identified as Serratia sp. strain DRY5 based on the Biolog GN carbon utilization profiles and partial 16S rDNA molecular phylogeny. Strain DRY5 produced 2.3 times the amount of Mo-blue than S. marcescens strain Dr.Y6, 23 times more than E. coli K12 and 7 times more than E. cloacae strain 48. Strain DRY5 required 37 degrees C and pH 7.0 for optimum molybdenum reduction. Carbon sources such as sucrose, maltose, glucose and glycerol, supported cellular growth and molybdate reduction after 24 hr of static incubation. The most optimum carbon source that supported reduction was sucrose at 1.0% (w/v). Ammonium sulphate, ammonium chloride, glutamic acid, cysteine, and valine supported growth and molybdate reduction with ammonium sulphate as the optimum nitrogen source at 0. 2% (w/v). Molybdate reduction was optimally supported by 30 mM molybdate. The optimum concentration of phosphate for molybdate reduction was 5 mM when molybdate concentration was fixed at 30 mM and molybdate reduction was totally inhibited at 100 mM phosphate. Mo-blue produced by this strain shows a unique characteristic absorption profile with a maximum peak at 865 nm and a shoulder at 700 nm, Dialysis tubing experiment showed that 95.42% of Mo-blue was found in the dialysis tubing suggesting that the molybdate reduction seen in this bacterium was catalyzed by enzyme(s). The characteristics of isolate DRY5 suggest that it would be useful in the bioremediation of molybdenum-containing waste

Chemopreventive potential of methanol extract of Dicranopteris linearis leaf on DMBA/croton oil-induced mouse skin carcinogenesis.

The present study was carried out to elucidate the chemopreventive potential of methanol extract of Dicranopteris linearis (MEDL) in a two-stage mouse skin carcinogenesis model due to the interrelated inflammation, oxidative stress and tumor promotion pathways. MEDL was prepared in a dose range of 30 to 300 mg/kg body weight. A total of 48 imprinting control region (ICR) female mice (6 to 8 weeks old) were randomly assorted into six groups. To induce skin tumor formation, a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) at 100 μg/100 μl was applied to the shaved dorsal region of mice, followed by repetitive administration of 1% croton oil, twice weekly for 15 weeks. Topical application of MEDL, 30 min prior to the croton oil application significantly reduced the tumor incidence to 12.5% in 300 mg/kg MEDL-treated group as compared to 87.5% in carcinogen control. The latency period of tumor formation was increased from sixth week in the carcinogen control to ninth and fifteenth weeks in 100 and 300 mg/kg MEDL-treated groups, respectively. The tumor burden of MEDL-treated groups (30, 100, and 300 mg/kg) were significantly lessen (5.67 ± 1.28, 5.00 ± 1.13, and 1.00 ± 0.13), as compared to carcinogen control (7.86 ± 2.37). The tumor volume was also significantly reduced from 9.00 ± 2.27 mm3 in carcinogen control to 3.70 ± 0.96, 2.39 ± 0.54 and 0.26 ± 0.03 mm3 in 30, 100 and 300 mg/kg MEDL-treated groups, respectively. In conclusion, the MEDL exhibited anti-carcinogenic effect in a dose-dependent manner, indicating its chemopreventive potential, which worth further study